Molecular Characterization of Class A-ESBLs, Class B-MBLs, Class C-AmpC, and Class D-OXA Carbapenemases in MDR Acinetobacter baumannii Clinical Isolates in a Tertiary Care Hospital, West Bengal, India

Background Acinetobacter calcoaceticus baumannii (ACB) complex has become a major concern nowadays because of its increasing involvement in several severe infections associated with catheter-related bloodstream and urinary tract infections, ventilator-associated pneumonia, cerebrospinal shunt-related meningitis, and wound infections. Multiple drug-resistant (MDR) ACB cases have been described with an increasing trend where at least it is resistant to a minimum of three antimicrobial groups. The mortality rate associated with A. baumannii is significantly higher than all Acinetobacter spp. isolates with the most prevalence seen in India and Thailand. The rapid spread of high resistance to most potent antimicrobial drugs is due to its ability to incorporate resistance determinants despite multifactorial reasons such as alteration in permeability of cell membrane by either losing expression of outer membrane porins or excess production of efflux pumps. This study aims to characterize resistance determinants responsible for MDR at the genetic level and emphasizes the use of genotyping in routine diagnosis as genotype analysis is reliable and valid. Methodology A total of 289 ACB complex clinical isolates were included in this study. The study for species-level identification of A. baumannii was conducted at the Department of Microbiology, IQ City Medical College Hospital, Durgapur, West Bengal. In addition, the detection of encoded genes associated with class A-extended spectrum beta-lactamases (i.e., CTX-M, KPC, SHV, and TEM genes), class B-metallo-β-lactamases (i.e., IMP, NDM, and VIM genes), Class C-AmpC cephalosporinase, and classD-OXA carbapenemases (i.e., blaOXA-10/11, blaOXA-24, blaOXA-48, blaOXA-58, blaOXA-143, and blaOXA-235 was done using real-time polymerase chain reaction. Results All 289 non-repetitive ACB complex clinical isolates were confirmed as A. baumannii, of which 277 (96%) isolates were MDR. There were no findings of blaCTX-M, blaKPC, blaSHV, blaTEM, blaIMP, blaVIM, blaAmpC, blaOXA-10/11, blaOXA-24, blaOXA-48, blaOXA-58, blaOXA-143, and blaOXA-235 genes in our study. However, there were four (1.44%) positive findings of the blaNDM gene. All MDR isolates (n = 277) were positive for the blaOXA-51 gene. In addition, blaOXA-23 was positive in 269 (97.12%) isolates. Conclusions The oxacillinase production corresponding to blaOXA-23 and blaOXA-51 were the most prevalent antibiotic resistance determinants among MDR A. baumannii in our study. Four (1.44%) isolates had the multiple genes blaOXA-51, blaOXA-23, and blaNDM that shows the coexistence of diverse genetic elements involved in MDR A. baumannii, resulting in total resistance except for a few potent drugs such as colistin and tigecycline. Genotyping is helpful in determining the contribution of the isolates in understanding their association with encoded genes, which, in turn, helps in designing effective surveillance and control strategies in the management of such MDR isolates.


Introduction
Acinetobacter calcoaceticus baumannii (ACB) complex has become a major concern nowadays because of its increasing involvement in several severe infections associated with catheter-related bloodstream and urinary tract infections, ventilator-associated pneumonia, cerebrospinal shunt-related meningitis, and wound infections [1]. It accounted for 1% to 2% of all bloodstream infections in a hospital-based study of developed countries, i.e., 52 hospitals located in the United States. They are typically associated with intravascular catheters (15.3%) and the respiratory tract (12.9%). Overall, 63% of the infections were associated with A. baumannii. The mortality rate associated with A. baumannii (36.8%) was significantly higher than those with A. nosocomialis (16.4%) and A. pittii (13.0%) [2]. Moreover, 1.2% to 87% of all Acinetobacter isolates were multiple drug resistant (MDR) with most prevalence seen in India and Thailand [3]. The MDR ACB is defined as isolates that provide resistance to a minimum of three antimicrobial groups [4]. This results in high rates of morbidity and mortality in the healthcare system due to its wider resistance to the most potent antimicrobial drugs. Moreover, there are reports of A. baumannii being acquired through the community [5].
The recently advanced whole-genome sequencing analysis has suggested the association of the rapid spread of MDR A. baumannii with the ability to incorporate resistance determinants [6][7][8]. Resistance to potent antibiotics in A. baumannii is primarily mediated by the genes that encode β-lactamase belonging to class A, B, and C AmpC cephalosporinase and D carbapenem-hydrolyzing β-lactamases despite multifactorial reasons such as alteration in the permeability of cell membrane by either losing expression of outer membrane porins or excess production of efflux pumps.
Our previous phenotypic study [4] clearly demonstrated that the ACB complex clinical isolates were resistant to the most potent drugs and further suggested its identification at the species level and association with resistance determinants at the genetic level. Therefore, this study was conducted to determine the incidence rate of A. baumannii from ACB complex clinical isolates and their resistance determinants responsible for βlactamase in association with MDR A. baumannii as genotype analysis is reliable and valid.

Bacterial isolates
The study for species-level identification of A. baumannii from previously phenotypically identified nonrepetitive clinical isolates of 289 ACB complex [4] was conducted at the Department of Microbiology, IQ City Medical College Hospital, Durgapur, West Bengal.

Bacterial DNA preparation and identification of A. baumannii
The commercially available spin column method of bacterial DNA isolation kit of GSure® Bacterial Genomic DNA Isolation Kit (GCC Biotech®, West Bengal, India) was used to prepare bacterial DNA isolates from a pure culture grown in peptone water and then kept at -20°C for further use.
The bacterial DNA extracts were identified for their species level, i.e., A. baumannii using real-time polymerase chain reaction (PCR) (CFX96™, Bio-Rad Laboratories, Inc.) for its further use in the genotypic analysis of certain antibiotic resistance determinants. A pair of forward and reverse primers that were specific to A. baumannii was used to amplify the target DNA. The hold stage was performed at the corresponding temperature of 50°C for five minutes. Consecutively, a corresponding temperature of 95°C was also applied for five minutes. In total, 45 cycles of continuous polymerization occurred at a corresponding temperature of 95°C for 15 seconds. Consecutively, a corresponding temperature of 60°C was also applied for 30 seconds.

Detection of encoded genes associated with drug resistance by realtime PCR
Multiplex PCR and uniplex PCR of certain antibiotic resistance determinants were analyzed as described below.

Multiplex Real-Time PCR Detection Assay
Multiplex real-time PCR was done to detect the class A group of β-lactamase, i.e., extended-spectrum (ESBLs) genes such as TEM, SHV, and CTX-M genes.
For this, 5 µL of extracted DNA template was added to a 20 µL reagent mixture (containing 12.5 µL master mix of Taq DNA polymerase, dNTPs mix, PCR buffer; 1 µL of internal control primer probe; 1 µL of internal control DNA; 3 µL of the respective primers, as shown in Table 1; and 2.5 µL of PCR-grade water). The initial denaturation of the above-mentioned genes was performed at a corresponding temperature of 95°C for 10 minutes. Thereafter, 40 cycles of further denaturation at a corresponding temperature of 95°C for 15 seconds. Annealing and extension were done at a corresponding temperature of 60°C for 30 seconds.  A second multiplex PCR was done to detect class B β-lactamase, i.e., metallo β-lactamase (MBLs) genes such as NDM, IMP, VIM, and KPC genes.
For this, a 5 µL extracted DNA template was added to a 20 µL reagent mixture (containing 12.5 µL master mix of Taq DNA polymerase, dNTPs mix, PCR buffer; 1 µL of internal control primer probe; 1 µL of internal control DNA, 4 µL of the respective primers, as shown in Table 1; and 1.5 µL of PCR-grade water). The initial denaturation of the above-mentioned genes was performed at a corresponding temperature of 95°C for 10 minutes. Thereafter, 45 cycles of further denaturation at a corresponding temperature of 95°C for five seconds. Annealing and extension were done at a corresponding temperature of 60°C for one minute.
A third multiplex PCR was done to detect the class D group of β-lactamase, i.e., carbapenem hydrolyzing (CHDLs) genes such as OXA-58, OXA-51, OXA-48, and OXA-23. The components and conditions for the thermocycle were identically performed as the above-mentioned second multiplex PCR except for the corresponding primers, as shown in Table 1.

Uniplex PCR
Uniplex PCR was done to detect the class C AmpC cephalosporinase gene and the CHDL genes such as OXA-24, OXA-143, and OXA-235.
For this, a 10 µL extracted DNA template was added to a 15 µL reagent mixture (containing 10 µL master mix of Taq DNA polymerase, dNTPs mix, PCR buffer, molecular grade water, and 5 µL of the respective primers, as shown in Table 1). The condition for thermocycle of the above-mentioned genes was Taq enzyme activation or the hold stage at a temperature of 95°C for about 15 minutes. Thereafter, 35 cycles of further denaturation at a corresponding temperature of 95°C for 20 seconds. Annealing was done at a corresponding temperature of 60°C for 20 seconds, followed by extension at a corresponding temperature of 72°C for 20 seconds.

Data analysis
A database for genotypic data collection and analysis was formed by using Microsoft Excel 2013 and descriptive analysis was done for the respective data.

Results
All 289 non-repetitive ACB complex clinical isolates were confirmed as A. baumannii by performing real-time PCR analysis (Figure 1). Out of the 289 confirmed A. baumannii, 277 (96%) isolates were MDR. These MDR A. baumannii isolates were used for further molecular analysis. The threshold cycle value is ≤40 for both the target as well as the internal control gene. The limit of detection was found to be DNA 10 copies/reaction.
Regarding the identification of the class A group of β-lactamase, i.e., ESBLs such as CTX-M, KPC, SHV, and TEM, there were no findings of bla CTX-M , bla KPC , bla SHV , and bla TEM genes in our study, as shown in Table 2.  Regarding the identification of class B β-lactamase, i.e., MBLs such as IMP, NDM, and VIM genes, there were no findings of bla IMP and bla VIM genes in our study, as shown in Table 2. However, there were four (1.44%) positive findings of the bla NDM gene, as shown in Table 2 and Figure 2. Regarding the identification of class C AmpC cephalosporinase, there were no findings of the AmpC (bla AmpC ) gene in our study, as shown in Table 2.
As shown in Table 2 and Figure 3, all the isolates (n = 277) were positive for the bla OXA-51 gene. In addition, bla OXA-23 was positive in 269 (97.12%) isolates, as shown in Table 2 and   Thus, the oxacillinase production corresponding to bla OXA-23 and bla OXA-51 were the most prevalent antibiotic resistance determinants among MDR A. baumannii. Four (1.44%) isolates had the multiple genes bla OXA-51 , bla  , and bla NDM that shows the coexistence of diverse genetic elements involved in MDR A. baumannii, resulting in total resistance except for a few potent drugs such as colistin and tigecycline.

Discussion
There was no significant variability among the data generated in the phenotypic and genotypic analyses but there was a comparative difference qualitatively. Both data have their associated advantages and disadvantages as bacterial phenotypic expression is affected by external physical factors. Therefore, the epidemiological typing was not enough by phenotypic analysis. Phenotypic tests are easy to perform and inexpensive compared to genotypic tests, whereas genotypic analysis is reliable and valid.
baumannii which shows that these genes can confer resistance to multiple potent antibiotics. According to several reports, the association of the insertion sequence ISAba1 element in conferring the carbapenemase activity of bla OXA-51 is important [5]. Therefore, the involvement of the bla OXA-51 gene alone in conferring resistance to multiple potent antibiotics is uncertain as there was no identification of the ISAba1 element through PCR in our study.
However, the detection of bla OXA-51 in all isolates supports the use of the bla OXA-51 gene as a surrogate marker of A. baumannii identification, as reported by different studies [9][10][11][12][13]. The bla OXA-51 gene has been shown to be widely associated with the ACB complex collected from various geographic regions [14][15][16][17][18]. This means that some of the members of the phenotypically identified ACB complex isolates possess the bla  [20].
The MBLs are less commonly identified in A. baumannii than oxacillinases, but their hydrolytic activities toward carbapenems except the monobactam aztreonam are significantly more potent (100 to 1,000 fold) [21]. In our study, there were no findings of bla VIM  substantiates the coexistence of diverse genetic elements involved in MDR A. baumannii, providing total resistance for potent drugs.
In our study, there were no findings of bla CTX-M , bla KPC , bla SHV , bla TEM , bla AmpC , bla OXA-235 , bla OXA-143 , bla OXA-58 , bla OXA-48 , bla OXA-24 , and bla OXA-10/11 genes. This study has limitations of a few small isolates and being a single-centered study. The association of insertion sequence with the overexpression of antibiotic resistance genes is yet to be explored in further studies. Moreover, the possibilities of diverse antibiotic resistance mechanisms such as alteration in the permeability of cell membrane, permeability by a decrease in the downregulation of outer membrane proteins, or upregulation of efflux pumps also exist.

Conclusions
This study shows that bla OXA-51 and bla OXA-23 are the most common antibiotic resistance determinants among MDR A. baumannii clinical isolates. The prevalence of multiple bla OXA-51 , bla OXA-23 , and bla NDM genes in isolates pose a threat to the healthcare community for outbreaks of total resistance to potent drugs and limit the therapeutic options. Moreover, there is an emergence of the bla NDM gene involved in MDR A.
baumannii clinical isolates that would show the serious problem of antimicrobial therapy. This shows the necessity of establishing relevant infection control practices or policies by respective stakeholders in response to the epidemiological data.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. IQ City Medical College & NH Hospital Institutional Ethics Committee (IQ City-IEC) issued approval IQMC/IEC/LTR/17/02/28 (28). Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.